The light-hyperresponsive high pigment-2dg mutation of tomato: alterations in the fruit metabolome

Overall metabolic modifications between fruit of light-hyperresponsive high-pigment (hp) tomato (Lycopersicon esculentum) mutant plants and isogenic nonmutant (wt) control plants were compared. Targeted metabolite analyses, as well as large-scale nontargeted mass spectrometry (MS)-based metabolite profiling, were used to phenotype the differences in fruit metabolite composition. Targeted high-performance liquid chromatography with photodiode array detection (HPLC-PDA) metabolite analyses showed higher levels of isoprenoids and phenolic compounds in hp-2dg fruit. Nontargeted GC-MS profiling of red fruits produced 25 volatile compounds that showed a 1.5-fold difference between the genotypes. Analyses of red fruits using HPLC coupled to high-resolution quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) in both ESI-positive and ESI-negative mode generated, respectively, 6168 and 5401 mass signals, of which 142 and 303 showed a twofold difference between the genotypes. hp-2dg fruits are characterized by overproduction of many metabolites, several of which are known for their antioxidant or photoprotective activities. These metabolites may now be more closely implicated as resources recruited by plants to respond to and manage light stress. The similarity in metabolic alterations in fruits of hp-1 and hp-2 mutant plants helps us to understand how hp mutations affect cellular processes.     

Phenotype of the tomato high pigment-2 mutant is caused by a mutation in the tomato homolog of DEETIOLATED1.

Tomato high pigment (hp) mutants are characterized by their exaggerated photoresponsiveness. Light-grown hp mutants display elevated levels of anthocyanins, are shorter and darker than wild-type plants, and have dark green immature fruits due to the overproduction of chlorophyll pigments. It has been proposed that HP genes encode negative regulators of phytochrome signal transduction. We have cloned the HP-2 gene and found that it encodes the tomato homolog of the nuclear protein DEETIOLATED1 (DET1) from Arabidopsis. Mutations in DET1 are known to result in constitutive deetiolation in darkness. In contrast to det1 mutants, tomato hp-2 mutants do not display any visible phenotypes in the dark but only very weak phenotypes, such as partial chloroplast development. Furthermore, whereas det1 mutations are epistatic to mutations in phytochrome genes, analysis of similar double mutants in tomato showed that manifestation of the phenotype of the hp-2 mutant is strictly dependent upon the presence of active phytochrome. Because only one DET1 gene is likely to be present in each of the two species, our data suggest that the phytochrome signaling pathways in which the corresponding proteins function are regulated differently in Arabidopsis and tomato.     

The tomato <Emphasis Type="Italic">dark green</Emphasis> mutation is a novel allele of the tomato homolog of the <Emphasis Type="Italic">DEETIOLATED1</Emphasis> gene

A comprehensive, multi-generation, allele test, carried out in this study, suggests that the tomato mutations dark-green (dg) and high pigment 2(j) (hp-2(j)) are allelic. The hp-2(j) mutant is caused by a mutation in the tomato homolog of the DEETIOLATED1 (DET1) gene, involved in the signal transduction cascade of light perception and morphogenesis. This suggestion is in agreement with the exaggerated photomorphogenic de-etiolation response of homozygous dg mutants grown under modulated light conditions. Sequence analysis of the DET1 gene was carried out in dg mutants representing two different lines, and revealed a single A-to-T base transversion in the second exon of the DET1 gene in comparison with the normal wild-type sequence. This transversion results in a conserved Asparagine(34)-to-Isoleucine(34) amino-acid substitution, and eliminates a recognition site for the AclI restriction endonuclease, present in the wild-type and in the other currently known tomato mutants at the DET1 locus. This polymorphism was used to develop a PCR-based DNA marker, which enables an early genotypic selection for breeding lycopene-rich tomatoes. Using this marker and sequence analysis we demonstrate that an identical base transversion also exists in dg mutants of the cultivar Manapal, in which the natural dg mutation was originally discovered. A linkage analysis, carried out in a F(2) population, shows a very strong linkage association between the DET1 locus of dg mutant plants and the photomorphogenic response of the seedlings, measured as hypocotyl length (12 < LOD Score < 13, R(2) = 51.1%). The results presented in this study strongly support the hypothesis that the tomato dg mutation is a novel allele of the tomato homolog of the DET1 gene.     

The Role of Polygalacturonase (PG) in Tomato Fruit Ripening and Effects of Divalent Metal Ions and Ethylene on PG Activity

It has been reported that PG is a key enzyme related to the tomato fruit ripening. In this study tomato fruits were harvested at the mature-green stage and stored at room temperature. The cell ultrastructure of pericarp tissue was observed at different ripening stages, and the effects of treatments with ethylene and calcium on PG activity and fruit ripening were examined. The object of this study is to elucidate the role of PG in regulation of tomato fruit ripening by ethylene and calcium. PG activity, was undetectable at mature-green stage, but it rose rapidly as fruif ripening. The rise in PG activity was coincided with the dechnmg of fruit firmness during ripening of tomato fruits. The observation of cell ultrastructure showed that the most of grana in chloroplast were lost and the mitochondrial cristae decreased as fruit ripening. Striking changes of cell wall structure was most noted, beginning with dissolution of the middle lamella and eventual disruption of primary cell wall. A similar pattern of changes of cell wall and chloroplast have been observed in pericarp tissue treated with PG extract. In fruits treated with calcium and other divalent metal ions atmature-green stage, the lycopene content and PG activity decreased dramatically. Ethylene application enhanced the formation of lycopene and PG activity. The inhibition of Ca2+ on PG ac ivity was removed by ethylene. Based on the above results, it was demonstrated that PG played a major role in ripening of tomato fruits, and suggested that the regulation of fruit ripening by ethylene and Ca2+ was all mediated by PG. PG induced the hydrolysis of cell wall and released the other hydrolytic enzymes, then effected the ripening processes follow up.     

Yield and post-harvest quality of tomato hybrids heterozygous at the loci alcobaça, old gold-crimson or high pigment

The effects of the fruit ripening mutant gene alcoba?a (alc) and color development mutants, old gold-crimson (ogc) and high pigment (hp), on yield and post-harvest quality of tomato fruits were investigated. Five tomato hybrids were obtained by crossing near isogenic lines with Flora-Dade background [Flora-Dade (alc+/alc+ ogc+/ogc+ hp+/hp+), TOM-559 (alc/alc ogc+/ogc+ hp+/hp+), TOM-591 (alc/alc ogc/ogc hp+/hp+), TOM-593 (alc/alc ogc+/ogc+ hp/hp), and TOM-589 (alc/alc ogc/ogc hp/hp)] with the pollen parent line Mospomorist (alc+/alc+ ogc+/ogc+ hp+/hp+). Hybrid fruit was harvested at the breaker stage and stored on shelves at 15oC and 60% relative humidity for 16 days, and then evaluated for firmness, development of red color, and carotenoid contents. The different genotypic combinations at the loci alc, ogc and hp had no effect on fruit yield. The alc+/alc hybrid genotype significantly increased fruit firmness and significantly delayed the development of red color in maturing fruit. Simultaneous usage of ogc+/ogc and hp+/hp promoted an increase in the red color and lycopene content of alc+/alc hybrids, but did not have any additional effect on fruit firmness.     

Relationships between fruit exocarp antioxidants in the tomato (Lycopersicon esculentum) high pigment-1 mutant during development

Development-dependent changes in fruit antioxidants were examined in the exocarp (epidermal and hypodermal tissues) of the monogenic recessive tomato (Lycopersicon esculentum L.) mutant high pigment (hp-1) and its wild-type parent 'Rutgers' grown under non-stress conditions in a greenhouse. The hp-1 mutant was chosen for this study because the reportedly higher lycopene and ascorbic acid (AsA) contents of the fruit may alter its tolerance to photooxidative stress. Throughout most of fruit development, reduced AsA concentrations in the exocarp of hp-1 were 1.5 to 2.0 times higher than in 'Rutgers', but total glutathione concentrations were similar in both genotypes. Only in ripe red fruit were reduced AsA and total glutathione concentrations lower in hp-1 than in 'Rutgers'. The redox ratios (reduced : reduced + oxidized) of AsA in hp-1 and 'Rutgers' exocarps were similar and usually > 0.9, however, the redox ratio of glutathione was lower in hp-1 than in 'Rutgers' throughout development. Lycopene concentrations in ripe red fruit were about 5 times higher in hp-1 than in 'Rutgers'. Large increases in the specific enzyme activities of superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) occurred during fruit development in both genotypes, with an inverse relationship between the activities of these enzymes and chlorophyll content. Glutathione reductase (EC 1.6.4.2) and MDHAR-specific activities were higher in hp-1 than 'Rutgers' only at the later stages of fruit development. Dehydroascorbate reductase (EC 1.8.5.1) activities, however, were usually higher in 'Rugters' than in hp-1. Catalase (CAT, EC 1.11.1.6) activities increased with fruit development until the fruit were orange/light red, when CAT was higher in 'Rutgers' than in hp-1, but then declined in the ripe red fruit of both genotypes. These results suggest that elevated AsA in the exocarp of hp-1 fruit early in fruit development may increase the tolerance of hp-1 fruit to photooxidative injury at that time, but the increasing activities of antioxidant enzymes appear to be developmentally associated with fruit ripening.     

Superoxide dismutase: A possible protective agent against sunscald in tomatoes (lycopersicon esculentum mill.)

Superoxide dismutase (SOD, EC 1.15.1.1) was concentrated from mature-green tomato fruits by gel chromatography. The enzyme was inhibited by cyanide but not by chloroform-ethanol, and appears to contain zinc and lesser amounts of copper. SOD-activity levels were high in immature green fruits, declined to a minimum in the mature-green and breaker stages known to be most susceptible to sunscald damage, increased again until the fruits were pink, and finally decreased through the red-ripe and overripe stages to the level of the mature-green fruit. When tolerance to sunscald damage was induced in mature-green fruits by controlled temperature treatment and samples of the fruits were challenged at various times during this process with a combined heat-and-light treatment known to cause sunscald, SOD activity was found to be inversely related to the susceptibility of the fruit to sunscald damage. It is suggested that superoxide is involved in sunscald injury to tomatoes and that tolerance is acquired through increases in SOD activity. Possibly SOD acts as a general protective agent against photodynamic damage to green tissues in plants that have become conditioned as the result of normal diurnal temperature fluctuations.     

Developmental changes in antioxidant metabolites, enzymes, and pigments in fruit exocarp of four tomato (Lycopersicon esculentum Mill.) genotypes: β-carotene, high pigment-1, ripening inhibitor, and ‘Rutgers’

In surface cell layers of fleshy fruit, antioxidants must limit photooxidative reactions that generate reactive oxygen species (ROS) in high light. Our objective was to measure changes in the concentrations of antioxidant metabolites and pigments, and the activities of enzymes of the Mehler-peroxidase, ascorbate-glutathione cycle in fruit exocarp tissue under non-stress conditions of the following fruit-specific tomato (Lycopersicon esculentum Mill.=Solanum lycopersicum) mutants and their parent: (1) beta-carotene (B), (2) high pigment (hp-1), (3) ripening inhibitor (rin), and (4) the nearly isogenic wild-type 'Rutgers'. Developmental variables included days after anthesis (DAA) and fruit surface color. The highest total ascorbic acid (AsA) concentration was in the exocarp of immature green fruit of hp-1, being 32% higher than 'Rutgers'. The hp-1 mutant also had the highest chlorophyll and total carotenoid concentrations, comprised mostly of lycopene in red ripe fruit; whereas, beta-carotene comprised 90% of the carotenoids in B. Although enzyme activities varied within genotype, they generally increased with development, then decreased as fruit maturity was reached, being coupled with AsA and glutathione (GSH) concentrations. In all mutants, dark-green (DG) exocarp had more chlorophyll and protein, higher concentrations of reduced AsA and GSH, and usually lower enzyme activities than light-green (LG) exocarp taken from the same fruit.     

Effects of ethylene on the susceptibility to Botrytis cinerea infection of different tomato genotypes

Ethylene at 10 and 100 μl/litre stimulated germ-tube elongation of Botrytis cinerea spores incubated within normal and non-ripening nor tomato fruits, but had little influence on the total percent of germination. Values of germ-tube length within the mature-green normal fruits and the mature-green or mature nor fruits were similar to those recorded within the normal mature fruits when held in air. Exposure of the normal and the mutant fruits to 100 μl/litre ethylene immediately after inoculation with B. cinerea insignificantly increased lesion development, but resulted in increased sporulation. When tomato fruits were exposed to ethylene for 3 days before inoculation a marked stimulatory effect on rot development was exhibited on the mature-green normal fruits but not on the nor mutant fruits. The results indicate that exogenous ethylene may directly stimulate germ tube growth of B. cinerea in both normal and mutant fruit, but that it may affect subsequent fungal growth indirectly, via stimulation of the ripening process, only in preclimacteric normal tomato fruit.     

Effect of the Calcium on Polygalacturonase (PG) Activity and Synthesis at Different Ripening Stages of Tomato Fruits

It has been reported that PG is a key enzyme related to the tomato fruit ripening and that the application of calcium can dramatically decrease the PG activity and delay the ripening of fruits. In this paper the effects of calcium treament at various ripening stages on the transformation of absorbed calcium, PG activity and PG synthesis in tomato fruits were studicd. According to the analysis of calcium by atomic absorption spectroscopy, it was shown that the soluble and total calcium contents in pericarp of fruits treated with calcium at mature-green stage were increased significantly, and that more soluble calcium was transformed into bound calcium. Both the absorption and transformation of calcium decreased in fruits treated with calcium at later stage of ripening. The inhibition of calcium on PG activity was most effective by treatment at mature-green stage, but less effective at later stage of ripening. One reason for the decrease of calcium inhibition was probably due to the decline of calcium absorption as fruit ripening. The polyacrylamide gel electrophoresis of PG showed that PG with a molecular weight of 46.7 kD was absent in mature-green fruits, and PG synthesis occurred only at the later stage of ripening. It seems that the earlier the treatment was done the more effective of the calcium inhibition of PG synthesis. Based on the above results, it was concluded that the PG plays a major role in ripening and senescence of tomato fruits, and both PG synthesis and its activity were inhibited by calcium. In order to delay the ripening and senescence of tomato fruits, the treatment with calcium should be done at mature-green stage.     

PG在番茄果实成熟中的作用及二价金属离子与乙烯对PG活性的影响

对采后番茄果实的电镜观察表明:当果实成熟衰老时,叶绿体数量减少,多数基粒结构丧失;成熟果实胞壁中胶层水解成中空的电子透明区,初生壁的纤丝也发生一定程度的水解,相邻细胞分离;外源 PG(多聚半乳糖醛酸酶)提取物处理绿熟期果实组织,也可引起胞壁结构和叶绿体发生与正常衰老相同的变化。Ca~(2+)、Mg~(2+)、Co~(2+)二价金属离子处理果实,可明显降低番茄红素含量和 PG 活性,延缓果实软化。外源乙烯处理果实,可促进番茄红素的形成,提高 PG活性,并能解除钙对 PG 活性的抑制。本文也对 PG 在乙烯和 Ca~(2+)调节果实成熟中的作用进行了讨论。     

钙对不同成熟期番茄果实的PG活性及其合成的影响

本文研究了钙处理不同成熟期番茄果实对果壁组织中钙含量与转化、多聚半乳糖醛酸酶(PG)活性与 PG 合成的影响。结果表明,钙处理绿熟期的番茄果实可使总钙和可溶性钙含量明显增加,并较多转化为结合钙;后期处理,进入和转化的钙都减少。同样,钙处理愈早,对果实 PG 活性的抑制愈强,绿熟期处理可完全抑制 PG 活性。凝胶电泳结合钌红染色,证明绿熟期果实无 PG,PG 是在果实成熟过程中新合成的。钙处理愈早,对 PG 合成的抑制愈强,绿熟期钙处理可完全抑制 PG 合成。